Nanoparticle solutions, kits, devices and methods of use thereof

This application is a § 371 national-stage application based on PCT/SG2021/050072, filed Feb. 11, 2021 which claims the benefit to Republic of Singapore Patent Application Number 10202001326W, filed Feb. 13, 2020, each of which is hereby incorporated by reference in its entirety.

The present invention relates, in general terms, to nanoparticle solutions, kits, devices and methods of use thereof. The present invention is suitable for use in quantifying bacterial cells in a sample.

Conventional methods used to detect and quantify bacteria are plate culturing, polymerase chain reaction (PCR), enzyme linked immunosorbent assay (ELISA) and chemical sensors based detection. However, such methods do not provide a quick readout of total bacteria count and requires a skilled person to work the method.

For example, the current industrial standard for bacteria detection is based on bacteria plate counting (plate culturing). This method takes about 2 to 7 days to obtain results, as the bacterial cells need to grow and multiply. Further, sample transfer to a laboratory is required, and the method cannot detect unculturable bacteria.

Accordingly, there is a need of a rapid on-site bacteria test for total bacteria count. There is a further need for such a test in the food and beverage (F&B) industry and many other industries, e.g. water industry, agriculture, aqua-culture, medical and pharmaceutical industries. There is also a need to have a reliable quantification method, which is easy to handle for use on site of the above mentioned industries.

It would be desirable to overcome or ameliorate at least one of the above-described problems, or at least to provide a useful alternative.

The present invention is predicated on the understanding that there is a lack of a simple yet rapid bacteria quantification test. Most current bacteria quantification tests require some sample treatment such as bacteria enrichment, or uses enzymatic reaction which can be easily affected by pH and temperature, or gives a delayed response. Accordingly, these bacteria quantification devices can be inaccurate as it is dependent on the sample preparation. For example, when nanoparticles are used to target bacterial cells, a source of error can come from the tendency of nanoparticles to self-aggregate, thus giving a false indication that bacterial cells are present. Another source of inaccuracy can be in the binding of nanoparticles to particulates other than bacterial cells. In addition, the quantification of live bacterial cells in contrast to dead bacterial cells should be distinguished as the presence of live bacterial cells is the cause of hygiene and health concerns while dead bacterial cells, if present after a sterilisation process, is generally not a health concern. The inventors have found that certain features as disclosed herein are beneficial for providing a bacterial cell quantification device which is rapid and at the same time accurate.

The present invention discloses a method of quantifying bacterial cells in a sample, comprising:

In some embodiments, the first ionic surfactant is selected from cetrimonium bromide (CTAB), cetrimonium chloride (CTAC) or sodium dodecyl sulfate (SDS).

Advantageously, the ionic surfactant provides a charged environment such that the nanoparticles are stabilised within the nanoparticle solution. This eliminates (or at least reduces) self-aggregation of the nanoparticles. It also reduces or eliminates non-specific binding to particulates which are not bacterial cells. The ionic surfactant also prevents non-specific background staining on the porous substrate.

In some embodiments, the first ionic surfactant in the first solution is at a concentration of at least 2 mg/mL.

In some embodiments, the plasmonic and/or fluorescent nanoparticle is selected from metallic nanoparticles, II-VI binary, ternary and quaternary semiconductor nanocrystals, IV-VI semiconductor nanocrystals, III-V semiconductor nanocrystals, I-V semiconductor nanocrystals, I-III-V semiconductor nanocrystals, group IV elemental semiconductor nanocrystals, Cuand Mndoped semiconductor nanocrystals, and their related core/shell structures thereof.

In some embodiments, the plasmonic and/or fluorescent nanoparticle is selected from gold nanoparticles, silver nanoparticles or CdSe/CdS core/shell nanorods.

In some embodiments, the nanoparticle is functionalised with an affinity probe selected from protein, sugar binding protein, peptide, or aptamer, such asII (GSII) lectin, BSA, and Antibac2 (AB2) aptamer. This is based on the affinity probe's specific recognition of commonalities in bacteria cells.

Advantageously, the presence of an affinity probe allows for rapid targeting of bacterial cells by the nanoparticle due to the low activation barrier. Further, affinity probe allows for a distinction between live and dead bacterial cells as the affinity interaction is reversible.

In some embodiments, a ratio of affinity probe to nanoparticle is about 1:1 to about 100:1.

In some embodiments, the nanoparticle is at a concentration of about 2 nM to about 30 nM.

In some embodiments, step (b) further comprises incubating the porous substrate with the first solution for at least 10 min.

In some embodiments, the nanoparticle is attachable to the bacterial cells via non-covalent interaction.

In some embodiments, the nanoparticle is attachable to the bacterial cells via electrostatic interaction, ionic bonding, Hydrogen bonding, Van der Waals interaction or a combination thereof.

In some embodiments, the second aqueous solution is a protein dissociation buffer such as glycine-HCl buffer at about pH 2.8 to about pH 3.5, or citric acid buffer at about pH 3.

In some embodiments, the second aqueous solution comprises a second surfactant selected from Tween-20, CTAB, CTAC, SDS.

Advantageously, the second solution when used to wash the bacterial cell sample allows for the preferential quantification of live bacterial cells over dead bacterial cells by disturbing the affinity interaction of the nanoparticle with dead bacterial cells, which is not as strong as with live bacterial cells.

In some embodiments, the second surfactant in the second aqueous solution is at a concentration of about 0.5 wt/wt % to about 1 wt/wt %.

In some embodiments, step (c) further comprises incubating the porous substrate with the second aqueous solution for at least 10 min.

In some embodiments, the colorimetric and/or fluorescence output emitted from the nanoparticle bound to the bacterial cells is measured or quantified by detecting a colorimetric and/or fluorescence output from the nanoparticles on the porous substrate.

In some embodiments, the colorimetric and/or fluorescence output emitted from the nanoparticle bound to the bacterial cells is detectable by eye.

In some embodiments, the method further comprises a step of quantifying the colorimetric and/or fluorescence output emitted from the nanoparticle bound to the bacterial cells via a detector.

In some embodiments, the method is for quantifying Gram-positive and/or Gram-negative bacterial cells in the sample.

In some embodiments, live bacterial cells in a sample is quantifiable from about 10cfu/mL to about 10cfu/mL.

In some embodiments, dead bacterial cells in a sample is quantifiable from more than about 10cfu/mL.

The present invention also discloses a device for quantifying bacterial cells in a sample in a liquid form, comprising:

In some embodiments, the porous substrate has a pore size of about 0.22 μm.

Advantageously, the porous substrate allows for the bacterial cells to be trapped on its surface while allowing other particulates to pass through. This provides for a high accuracy. Further, the aqueous solutions are allowed to flow through unhindered, thus shortening the time for quantification.

In some embodiments, the device further comprises a detector for quantifying the colorimetric and/or fluorescence output emitted from the nanoparticle bound to the bacterial cells.

In some embodiments, the device is for quantifying Gram-positive and/or Gram-negative bacterial cells in the sample.

In some embodiments, the limit of detection for live bacterial cells in a sample is about 10cfu/mL.

In some embodiments, the limit of detection for dead bacterial cells in a sample is about 10cfu/mL.

The present invention discloses an aqueous nanoparticle solution, comprising:

The present invention also discloses a kit, comprising:

The technical challenges in relation to the present invention includes an understanding of the affinity probes' biochemical properties, charges, functional groups, as well as the membrane's surface properties, in order to derive a suitable formulation to prevent the non-specific background staining, and at the same time retain the affinity interaction between the particles and the bacteria cells. In addition to the proper selection of suitable surfactants, optimizing the surfactant concentration can also be important to minimize the non-specific staining without introducing crystallization of surfactant (due to too high concentration of surfactant).

Accordingly, the present invention discloses a method of quantifying bacterial cells in a sample, comprising:

In some embodiments, the method is for quantifying live bacterial cells in a sample. Accordingly, the method is able to distinguish between live bacterial cells and dead bacterial cells. For example, as shown in, the method can detect both Gram-positive (e.g.) and Gram-negative (e.g.) bacteria. Further, as shown in, the method preferentially detects live cells. Dead cells at a high concentration of 10CFU/ml do not show detection signal.

As used herein, ‘bacterial cell quantification’ refers to the measurement or determination of the total number of bacterial cells in a sample. In this regard, there is no distinction regarding the species/type of bacterial cells. In some embodiments, the total number of bacterial cells is the total number of live bacterial cells. The sample can be an unadulterated sample or unprocessed sample. The sample can be a processed sample, for example a diluted sample or a concentrated/neat sample.

As used herein, “flowing” refers to moving the sample continuously as a stream through the porous substrate. In some embodiments, the sample (and/or the solutions) is passed through the porous substrate. The sample contacts the porous substrate at one surface, permeates the porous substrate and exits the porous substrate via an opposite surface. To this end, the sample is in a liquid form.

In some embodiments, the method of quantifying bacterial cells in a sample comprises:

The passing of the sample in a liquid form through a porous substrate serves to trap or retain bacterial cells on the surface of the substrate. In some embodiments, the sample is filtered through the porous substrate. This step allows for the separation of bacterial cells from the other contents in the sample which can result in a high background noise.

As used herein, ‘aqueous solution’ refers to a water based solvent or solvent system, and which comprises of mainly water. Such solvents can be either polar or non-polar, and/or either protic or aprotic. Solvent systems refer to combinations of solvents which resulting in a final single phase. Both ‘solvents’ and ‘solvent systems’ can include, and is not limited to, pentane, cyclopentane, hexane, cyclohexane, benzene, toluene, dioxane, chloroform, diethylether, dichloromethane, tetrahydrofuran, ethyl acetate, acetone, dimethylformamide, acetonitrile, dimethyl sulfoxide, nitromethane, propylene carbonate, formic acid, butanol, isopropanol, propanol, ethanol, methanol, acetic acid, ethylene glycol, diethylene glycol or water. Water based solvent or solvent systems can also include dissolved ions, salts and molecules such as amino acids, proteins, sugars and phospholipids. Such salts may be, but not limited to, sodium chloride, potassium chloride, ammonium acetate, magnesium acetate, magnesium chloride, magnesium sulfate, potassium acetate, potassium chloride, sodium acetate, sodium citrate, zinc chloride, HEPES sodium, calcium chloride, ferric nitrate, sodium bicarbonate, potassium phosphate and sodium phosphate. As such, biological fluids, physiological solutions and culture medium also fall within this definition. In most embodiments, the aqueous solution is water. In some embodiments, the aqueous solution is deionised water. In some embodiments, the aqueous solution is Millipore water.

An aqueous solution is advantageously used in the present disclosure. In particular, water is used. Water is a green solvent and can be manipulated by the user with ease, without concern of toxicity and change in concentration due to evaporation. Water also does not adversely impact the porous substrate.

‘Nanoparticle’ refers to a nano-object with all three external dimensions in the nanoscale. For example, nanoparticle can be particles between 1 nm and 990 nm in size. In an embodiment, the nanoparticle is less than about 100 nm in diameter. Because of their nano-size, properties which are different from the physical bulk properties are manifested.